The long term goal of our research is to understand the molecular mechanisms driving the propagation and termination of protein kinase C (PKC) signaling in response to agonists in cells. PKC has been in the spotlight since the discovery three decades ago that it is activated by the lipid second messenger, diacylglycerol. Despite PKC's enduring stage presence and tremendous advances in understanding the enzymology and regulation of this key protein, understanding the function of PKC in biology is still under intense pursuit. We have previously focused on understanding the molecular mechanisms of PKC as a first step in understanding how this key protein functions in the cell. Under the auspices of the MERIT Award, we ventured away from our biochemical studies and dove into the cell, asking the guestion: what does protein kinase C do in the cell? We developed novel genetically-encoded reporters that allowed us to simultaneously visualize PKC activity and second messenger production in real time in live cells. In the next funding period, we propose to take this new technology to the next level and develop reporters that will allow us to simultaneously image two or more kinase activities at in the same cell. This will allow us to examine the interplay between different signaling pathways, feedback mechanisms, and identify regulatory inputs that our previous biochemical studies could not address. The three major questions we ask are: 1] how does PKC impact other signaling pathways? 2] how do phosphatases control signal amplitude and signal termination? and 3] how are atypical PKC isozymes regulated in cells? Thus, in the next funding period, we aim to understand the cellular mechanisms of signal propagation, signal termination, and signal cross-talk using novel fluorescence technologies to 'spy' on cell signaling.